Modular engineering of L-tyrosine production in Escherichia coli

Abstract

Efficient biosynthesis of L-tyrosine from glucose is necessary to make biological production economically viable. To this end, we designed and constructed a modular biosynthetic pathway for L-tyrosine production in E. coli MG1655 by encoding the enzymes for converting erythrose-4-phosphate (E4P) and phosphoenolpyruvate (PEP) to L-tyrosine on two plasmids. Rational engineering to improve L-tyrosine production and to identify pathway bottlenecks was directed by targeted proteomics and metabolite profiling. The bottlenecks in the pathway were relieved by modifications in plasmid copy numbers, promoter strength, gene codon usage, and the placement of genes in operons. One major bottleneck was due to the bifunctional activities of quinate/shikimate dehydrogenase (YdiB), which caused accumulation of the intermediates dehydroquinate (DHQ) and dehydroshikimate (DHS) and the side product quinate; this bottleneck was relieved by replacing YdiB with its paralog AroE, resulting in the production of over 700 mg/liter of shikimate. Another bottleneck in shikimate production, due to low expression of the dehydroquinate synthase (AroB), was alleviated by optimizing the first 15 codons of the gene. Shikimate conversion to L-tyrosine was improved by replacing the shikimate kinase AroK with its isozyme, AroL, which effectively consumed all intermediates formed in the first half of the pathway. Guided by the protein and metabolite measurements, the best producer, consisting of two medium-copy-number, dual-operon plasmids, was optimized to produce >2 g/liter L-tyrosine at 80% of the theoretical yield. This work demonstrates the utility of targeted proteomics and metabolite profiling in pathway construction and optimization, which should be applicable to other metabolic pathways.

Fig 1

(A) The biosynthetic pathway of l-tyrosine (TYR) in E. coli from glucose. X5P, xylulose 5-phosphate; PYR, pyruvate; EPSP, 5-enolpyruvoylshikimate 3-phosphate; CHA, chorismate; PPA, prephenate; HPP, 4-hydroxyphenlypyruvate; l-Glu, glutamic acid; and α-KG, α-ketoglutarate. The enzymes (in boldface) are as follows: PpsA, phosphoenolpyruvate synthase; TktA, transketolase A; AroG, DAHP synthase; AroB, DHQ synthase; AroD, DHQ dehydratase; YdiB, quinate/shikimate dehydrogenase; AroE, shikimate dehydrogenase; AroK/L, shikimate kinase I/II; AroA, EPSP synthase; AroC, chorismate synthase; TyrA, chorismate mutase/prephenate dehydrogenase; and TyrB, tyrosine aminotransferase. QUIN and gallic acid (GA) are side products. QUIN is formed by YdiB from DHQ (18), while GA is formed by AroE from DHS (18, 30). The dashed lines indicate where feedback inhibitions occur. Allosteric regulation of AroG and TyrA were removed in this study by employing their respective feedback-resistant mutants, AroG* (D146N) and TyrA* (M53I;A354V), respectively. (B) Structures of the initial modules, S0 and Y0, for production of shikimate and l-tyrosine, respectively. The open blocks indicate the origins of replication, the shaded arrows represent the genes, and the angled arrows indicate the promoters. Note that for each operon, the genes are placed in the reverse order relative to the reaction pathway. Using the shikimate module as an example, ydiB, which catalyzes the last step in the formation of shikimate, was placed next to the promoter, and so on. (C) SRM analysis of the protein production levels from S0 and Y0 in strain A when induced or uninduced with IPTG. The protein levels shown are ratios relative to the uninduced levels.

Fig 2

(A) Stepwise improvements of the shikimate module by changing the origin of replication from pSC101 to pBBR1 and the promoter from P_LtetO-1 to P_lac-UV5 (S1), followed by codon optimization of aroB (S2), replacement of ydiB with aroE (S3), and inserting a second promoter P_LtetO-1 5′ of aroG* (S4). In S5, a combination of the rrnB terminator T1 (large T) and P_trc was used to substitute P_LtetO-1 5′ of aroG*, which resulted in significant reductions in protein and shikimate production. (B) The SRM results indicate the relative levels of TktA through YdiB/AroE as a consequence of the various modifications to the shikimate module. All cultures were performed under the conditions described in Table 3.

Fig 3

(A) Stepwise improvements of the tyrosine module by replacement of aroK with aroL (Y1) and inserting a second promoter, either P_LtetO-1 (Y2) or a combination of the rrnB terminator T1 (large T) and P_trc (Y3), 5′ of aroA. (B) The SRM results indicate the relative levels of TyrB through AroL as a consequence of the various modifications to the tyrosine module. All cultures were performed under the conditions described in Table 3.